1. Field of the Invention
The present invention relates to a method of forming a planar bilayer membrane with a desired size on a substrate in a liquid phase, using a membrane-forming substance such as lipid or protein, under mild conditions and according to simple procedures.
2. Related Background Art
It is well known that biomembrane is comprised of a lipid bilayer membrane as a basic structure, and fulfill a great variety of functions because of the various membrane proteins present in the membrane in a mixed state. If an artificial membrane can be reconstructed to mimick a biomembrane, it can be utilized for the filtration or concentration of useful substances by controlling, for example, the permeability to substances. It can also be used for the adsorption and combination of a trace biotic substances by utilizing the affinity for the biotic substances. For example, enzyme or antibody sensors are developed which are capable of detecting substances by retaining enzymes or antibodies in the membrane and by measuring the membrane potential or change in membrane resistance occuring when they have attached to a specific substrate or antigen.
To carry out development of such functional membranes, required first of all technique is required for constructing a stable planar bilayer membrane of lipid. It is further preferred that protein can be introduced into the membrane under mild conditions. Hitherto known methods of preparing a planar membrane include the black membrane process and the Langmuir-Blodgett process (LB process).
The black membrane process utilized the phenomenon that, when a thin sheet made of plastics or the like in which minute holes of about 100 .mu.m in diameter are made is immersed in an aqueous solution and a lipid dissolved in an organic solvent is applied on the minute holes, the solvent moves over the peripheries of the minute holes, so that a lipid bilayer membrane is formed at the central area.
The LB process is a process by which a monolayer, membrane or a built-up membrane thereof is formed by utilizing the phenomenon that, in molecules having a hydrophilic part and a hydrophobic part therein, or molecules having the parts that show a remarkable difference in the hydrophilic characteristics from each other the molecules form a monolayer with hydrophilic groups facing down on a water surface. These membranes are usually prepared using a Langmuir's water tank. This water tank is provided with a partition plate so that a molecular layer dispersed on the gas-liquid interface may not loosely disperse on the aqueous phase. Thus the dispersing area of the molecular layer spread can be restricted and hence the assembled state of the molecular layer can be restricted, and a surface pressure is obtained depending on the assembled state. The partition plate is moved so that the dispersing area may be reduced, thereby gradually increasing the surface pressure, and thus a surface pressure of the molecular layer can be set. Then, while maintaining the surface pressure, a clean substrate is gently moved upward and downward in the vertical direction or the substrate is horizontally laid on the molecular surface, so that the molecules can adhere to and be transferred on the substrate.
The membrane-forming substances such as protein and lipid are not necessarily stable under physical and chemical conditions in the conventional procedures for preparing planar membranes. The protein exhibits its function only when the polypeptide chain keeps a specific higher order structure, and tends to be denatured or inactivated as a result of the destruction of the higher order structure by interfacial tension, organic solvents or the like. The lipid also tends to undergo oxidation in the presence of oxygen.
In the black membrane process, in which an organic solvent is used in the procedure for preparing a membrane, it is commonly difficult to simultaneously introduce protein into the membrane. The organic solvent also inevitably remains in the membrane produced, and hence there is the difficulty of inactivation even when the protein is introduced according to, for example, proteoliposome fusion after the preparation of a membrane.
In the LB process, the membrane-forming substance must be previously dispersed as a monolayer membrane on the gas-liquid interface, so that the protein may sometimes be denatured by interfacial tension. There is also a possibility of the oxidation of lipid because of its contact with a gaseous phase.
Thus, the conventional methods of preparing the planar membrane, which have taken no account of the physical and chemical stability of molecules, have sometimes caused defects in the membrane particularly when protein or lipid originating from organisms is used, resulting in no exhibition of the expected functions. It is therefore sought to prepare the membrane under milder conditions.
The conventional methods of preparing the planar membrane further have the following disadvantages. In the black membrane process, the area and shape of the membrane that can be prepared are so extremely limited that it is impossible to prepare a membrane with a diameter of more than 1 mm. In preparing the membrane, a skilled operation is also required. Thus, they are not suitable for their industrial application.
The LB process is actually subject to various limitations based on, e.g., the shape of apparatus, in regard to the area and shape of the membrane, but is free from any limitations in principles. As previously mentioned, however, in the LB process the building-up of membranes in the molecular level is carried out by mechanical action, and hence delicate precision is required in controlling the surface pressure and moving the substrate. Moreover, because of use of the monolayer membrane dispersed on the gas-liquid interface, even a slight vibration of the water surface and inclusion of a trace of dust floating in the air may give defects to the membrane. Hence, in order to obtain an LB membrane with good quality, an investment must be made for the installation of an anti-vibration apparatus, a clean room, and so forth.